The management of malignant gliomas remains one of the most challenging issues in neuroscience. Gene therapy strategies currently used in clinical trials employ viral vectors to deliver therapeutic transgenes directly to normal and tumor cells within the central nervous system. Viral vectors, however, have a number of theoretical and practical limitations. Embryonic stem cells (ESC) are totipotent cells with unlimited proliferative capacity, and, unlike other cell types, can be permanently and precisely genetically modified without the use of viral vectors. We have published a protocol for generating highly pure populations of transgenic astrocytes from mouse ESC, while minimizing contamination by undifferentiated ES cells. We have demonstrated that we can generate a pure population of transgenic ESC-derived astrocytes with a highly regulated, robust tetracycline-inducible expression of transgenes. We have demonstrated that ESC-derived astrocytes conditionally expressing pro-apoptotic genes induce apoptosis in vitro and in vivo in malignant glioma cells. Finally, we have shown that ESC-derived astrocytes conditionally expressing transgenes have migratory capacities in vitro and in vivo. Recent data show that the melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL- 24) gene induces apoptosis in a variety of tumor cell lines, including gliomas, while sparing normal cells. We have engineered ESC-derived astrocytes to conditionally express mda-7/IL-24. It is our hypothesis that ESC-derived astrocytes expressing mda-7/IL-24 may represent an effective and novel approach for treatment of human malignant gliomas. In this proposal, we will test our hypothesis in the following specific aims. 1) To determine the behavior of mouse ESC-derived astrocytes conditionally expressing mda-7/IL-24 in situ after grafting into the mouse brain; 2) To determine the pro-apoptotic effects of ESC-derived astrocytes conditionally expressing mda-7/IL-24 in vitro, in vivo, and in situ on human malignant glioma; 3) To investigate the effects of transgenic mda-7/IL-24 to enhance prop-apoptotic effects of conventional ionizing radiation (IR) and those of transgenic TRAIL. The effective treatment of malignant gliomas will depend on the development of therapeutic strategies that can eradicate residual tumor cells left behind after sophisticated surgical resection or stereotactically delivered high dose radiation. Therefore, the migratory characteristic of ESC-derived astrocytes combined with the capability of carrying transgenes tightly regulated by external control, makes ESC-mediated gene delivery a promising new treatment modality for human malignant gliomas.